Micropatterns of Chemisorbed Cell Adhesion-Repellent Films Using Oxygen Plasma Etching and Elastomeric Masks Anna Tourovskaia, † Thomas Barber, ‡ Bronwyn T. Wickes, § Danny Hirdes, † Boris Grin, † David G. Castner, †,§ Kevin E. Healy, ‡ and Albert Folch* ,† Departments of Bioengineering and Chemical Engineering, University of Washington, Seattle, Washington 98195; and Departments of Bioengineering and Materials Science and Engineering, University of California at Berkeley, Berkeley, California 94720 Received November 4, 2002. In Final Form: February 3, 2003 Cellular micropatterning has become an important tool to precisely design cell-to-substrate attachment for cell biology studies, tissue engineering, cell-based biosensors, biological assays, and drug screens. This paper describes a new technique for micropatterning of cells that is based on the use of oxygen plasma as a patterning tool. The technique consists of (1) homogeneously grafting a glass substrate with a protein- repellent interpenetrating polymeric network (IPN) of poly(acrylamide) and poly(ethyleneglycol) [P(AAm- co-EG)] prepared with commercially available reagents and (2) selectively removing this coating using oxygen plasma. We use elastomeric stencils (i.e. self-sealing membranes with through-holes) and microchannels as removable masks for the selective oxygen plasma etch of the IPN areas that are not protected by the mask. The stencil or microchannels are peeled off to reveal cell-adhesive regions separated by the nonadhesive coating. Our method offers a convenient way of patterning a robust protein-repelling material, allows for independently controlling the chemistry of the regions reserved for cell attachment, and can be used to create coculture systems. Introduction Numerous microfabrication approaches have been devised to control and modulate cell-cell, cell-substrate, and cell-medium interactions on a micrometer scale. 1 These techniques reproduce in vivo microenvironments better than homogeneous cultures and offer a great potential not only for studies of molecular and cell biology but also for engineering tissue replacements and for developing cell-based sensors and drug screens. Cell micropatterning approaches largely utilize one of two strategies to deposit cells on designated areas of the cell culture substrates (for a review, see ref 1): (1) selective cell attachment is guided by differential adhesiveness of the substrate, or (2) cell attachment to a homogeneously adhesive substrate is blocked in selected areas with a removable physical barrier. Recently, soft lithographic methods have been developed to selectively deposit cell/ protein-repellent/adhesive coatings from solution, 2,3 by microcontact printing, 4-7 and to deposit cells directly 7-11 using microfluidic and micromolding techniques (for a review, see ref 1). Surface chemistry has been a powerful tool to produce protein-repellent coatings and to dictate protein adsorption and cell attachment onto artificial materials 12 (for a review, see ref 13). The time span within which these materials can maintain the spatial distribu- tion of cells varies widelysfrom several hours to 60 days. 1,14 The most successful approaches to engineering long-term selective cell adhesion/spreading have utilized ethyleneg- lycol-terminated self-assembled monolayers (SAMs), 4 polymeric thin films containing poly(acrylamide) and poly- (ethyleneglycol) (PEG), 15-17 polyacrylamide, 14 plasma- polymerized poly(ethyleneglycol)-like films (“tetragly- me”), 18,19 or a commercial copolymer of poly(ethyleneoxide) and poly(propyleneoxide) (Pluronic). 3,20 Among those techniques, the grafted interpenetrating network of poly- (acrylamide-co-ethyleneglycol) [P(AAm-co-EG) IPN] de- veloped by Healy and co-workers 15-17 is especially at- tractive because (1) it resists nonspecific protein adsorption and has maintained cell patterns for the longest time † Department of Bioengineering, University of Washington. ‡ University of California at Berkeley. § Department of Chemical Engineering, University of Wash- ington. (1) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227-256. (2) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. (3) Liu, V. A.; Jastromb, W. E.; Bhatia, S. N. J. Biomed. Mater. Res. 2002, 60, 126-134. (4) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305-313. (5) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (6) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (7) Tien, J.; Nelson, C. M.; Chen, C. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1758-1762. (8) Folch, A.; Jo, B.-H.; Beebe, D.; Toner, M. J. Biomed. Mater. Res. 2000, 52, 346-353. (9) Folch, A.; Ayon, A.; Hurtado, O.; Schmidt, M. A.; Toner, M. J. Biomech. Eng. 1999, 121, 28. (10) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408-2413. (11) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811-7819. (12) Drumheller, P. D.; Hubbell, J. A. In The Biomedical Engineering Handbook; Bronzino, J. D., Ed.; CRC Press: Boca Raton, FL, 1995; pp 1583-1596. (13) Mrksich, M. Cell. Mol. Life Sci. 1998, 54, 653-662. (14) Nelson, C.; Raghavan, S.; Tan, J.; Chen, C. Langmuir 2003, 19, 1493-1499. (15) Bearinger, J. P.; Castner, D. G.; Golledge, S. L.; Rezania, A.; Hubchak, S.; Healy, K. E. Langmuir 1997, 13, 5175. (16) Bearinger, J. P.; Healy, K. E. J. Dent. Res. 1997, 76, 2948. (17) Thomas, C. H.; Lhoest, J. B.; Castner, D. G.; McFarland, C. D.; Healy, K. E. J. Biomech. Eng. 1999, 121, 40. (18) Lopez, G. P.; Ratner, B. D.; Tidwell, C. D.; Haycox, C. L.; Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1992, 26, 415-439. (19) Pan, Y. V.; McDevitt, T. C.; Kim, T. K.; Leach-Scampavia, D.; Stayton, P. S.; Denton, D. D.; Ratner, B. D. Plasmas Polym. 2002, 7, 171-183. (20) Neff, J. A.; Tresco, P. A.; Caldwell, K. D. Biomaterials 1999, 20, 2377-2393. 4754 Langmuir 2003, 19, 4754-4764 10.1021/la0267948 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/26/2003